Generation of nanodecoys
Nanodecoys were derived from LSCs or HEK293 cells (ATCC CRL-1573) by an extruder (LiposoFast LF-50, AVESTIN). Cells were collected and suspended in PBS at a concentration of 5 × 106 cellen ml-1. A large volume of cells could be extruded immediately or stored at −80 °C until ready. The cells were passed through the extruder twice through 5 μm, 1 μm and 400 nm pore-sized polycarbonate membrane filters (Avanti Polar Lipids) sequentially. The resulting nanodecoys were purified and concentrated using an ultrafiltration centrifuge tube (100 kDa Mw cut-off; Millipore) and centrifuged at 4,500 g for 10 min and washed with PBS. The size and concentration of nanodecoys were measured using an nanoparticle tracking analysis system (NanoSight, Malvern Panalytical). Nanodecoys were stored at 4 °C for 1 week or placed in long-term storage at −80 °C. The ACE2 receptors on the nanodecoys were detected using immunoblot, immunostaining, flow cytometry and TEM with immunogold labelling.
ACE2 analysis using ELISA
LSC and HEK293 cells (5 × 106 of each) were collected and 1010 of LSC-nanodecoys and HEK293-nanodecoys were prepared. They were analysed with an ACE2 ELISA kit (ab235649, Abcam) according to the manufacturer’s instructions.
In vitro internalization experiments of nanodecoys
Human macrophage primary cells and LSCs (104 cellen ml-1) were seeded in four-well culture chamber slides (Thermo Fisher Scientific). Nanodecoys (1 × 106 cellen ml-1) were then labelled with DiD and incubated with macrophages or LSCs alone, and also with a co-culture of both (1:1) to mimic the in vivo microenvironment. After 4 h of incubation, free nanodecoys were removed by three washes with 1× PBS. Cells were fixed with 4% paraformaldehyde (PFA) before immunocytochemistry staining with markers for macrophage (CD4; 12-0041-82, Invitrogen) and LSC (CD90; 11-0909-42, Invitrogen) and imaged with an Olympus FluoView confocal microscope. In addition, to quantify the internalization rate of nanodecoys by the different cell types, cells and nanodecoys were cultured in a T75 flask as previously described and collected for flow cytometry analysis (CytoFlex, Beckman Coulter).
In vitro spike S1 neutralization experiments of nanodecoys
Recombinant spike S1 (40591-V08H, Sino Biological; 10 ng ml-1, Mw 76.5 kDa) was added to nanodecoys at different concentrations (5 × 109, 1 × 109, 2 × 108, 4 × 107, 8 × 106, 1.6 × 106 en 3.2 × 105) and incubated for 3 h. After that, the unbound spike S1 was removed by ultracentrifugation (100 kDa). Spike S1 before and after binding to nanodecoys was determined with an ELISA kit (SARS-CoV-2 Spike ELISA Kit, Sino Biological) according to the manufacturer’s protocol. To study the neutralization of spike S1 with nanodecoys in primary lung-derived cells (LSCs), spike S1 was first labelled with NHS-Rhodamine (46406, Thermo Fisher Scientific) according to the manufacturer’s instructions. The rhodamine B (RhB)-spike S1 (100 ng) was first incubated with LSCs (2 × 104) in four-well slides for 1 h and washed with PBS three times. DiD-labelled nanodecoys (2 × 107) were then added and incubated for another 4 h. Cells were washed and fixed using 4% PFA before staining with Alexa Fluor 488 phalloidin (A12379, Invitrogen). Cells were imaged using an Olympus FluoView confocal microscope.
Generation of SARS-CoV-2-mimicking virus
Spike S1 (40591-V08H, Sino Biological) was conjugated to lentivirus (Cellomics Technology) to create a SARS-CoV-2 mimic. The His-tagged spike S1 was linked to Ni-NTA through chemical interaction. NTA with a mercapto group (N-[Nα,Nα-bis(carboxymethyl)-l-lysine]-12-mercaptododecanamide) was first reacted with 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC) to give NTA-SMCC and was then added to the lentivirus. The NTA groups were conjugated to the lentivirus through the –NH2 group on the lentivirus and the N-hydroxysuccinimide ester on NTA-SMCC. The free NTA-SMCC was removed by centrifugation using an ultrafiltration tube (100 kDa Mw cut-off; Millipore) to give SARS-CoV-2-mimicking virus (spike S1-lentivirus). The successful conjugation of spike S1 on lentivirus was confirmed using TEM. Briefly, SARS-CoV-2 mimics were incubated with anti-spike S1 antibodies overnight at 4 °C. Free antibodies were removed using an ultrafiltration tube (100 kDa Mw cut-off; Millipore) and washed with PBS three times. The spike S1 on the SARS-CoV-2 mimics was labelled with immunogold (10 nm) antibodies and negatively stained for TEM visualization. The conjugation efficiency of spike S1 on lentivirus was determined using ELISA (SARS-COV-2 Spike ELISA Kit, Sino Biological) according to the manufacturer’s protocol.
SARS-CoV-2-mimicking virus in cells
LSC cells (104 cellen ml-1) were seeded in eight-well culture chamber slides (Thermo Fisher Scientific) and allowed to adhere for 24 h. SARS-CoV-2 mimics (104 TU ml-1) were added into the eight-well slides and incubated for 4 h. Next, LSC cells were washed with PBS twice to remove non-internalized SARS-CoV-2 mimics and stained with 100 μM Lyso Dye (Invitrogen, green) at 37 °C for 30 min. Subsequently, slides were mounted with ProLong Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) and imaged on an Olympus FluoView FV3000 confocal laser scanning microscope with an Olympus UPlanSAPO ×60 objective (1-U2B832).
SARS-CoV-2 mimic neutralization experiment
Nanodecoys were first labelled with DiI. Next, 200 μl of SARS-CoV-2 mimic (5 × 105) in pH 9.6 coating buffer was added to each well of 96-well plates and incubated at 4 °C overnight for coating. In addition, lentiviruses without spike S1 were also coated to the plates as a control. Following the incubation, the protein solution was removed, and the wells were washed with 1× PBS. To study binding, plates were incubated with DiI-labelled nanodecoys at concentrations of 1 × 104, 2 × 104, 4 × 104, 8 × 104, 1.6 × 105, 3.2 × 105, 6.4 × 105 en 1.28 × 106 for 2 h at room temperature. The plates were then rinsed with 1× PBS three times, and fluorescent intensities were determined with a microplate reader (Molecular Devices).
Interaction of SARS-CoV-2 mimic with LSCs was assessed by ICC and flow cytometry. RhB-NHS was first reactivated with NTA-tagged lentivirus and then modified with S1 protein to synthesize RhB-labelled SARS-CoV-2 mimics. LSCs (104 cellen ml-1) were seeded in four-well culture chamber slides. RhB-labelled lentivirus (104 TU ml-1), RhB-labelled SARS-CoV-2 mimic (104 TU ml-1), RhB-labelled SARS-CoV-2 mimic (104 TU ml-1) + LSC-nanodecoys (105), and RhB-labelled SARS-CoV-2 mimic (104 TU ml-1) + HEK-nanodecoys (105) were incubated with LSCs, respectively. After 4 h of incubation, free SARS-CoV-2 mimics were removed and washed with PBS three times. Cells were fixed with 4% PFA, stained for LSC markers (fluorescein isothiocyanate (FITC)-labelled CD90; 11-0909-42, Invitrogen), and imaged with an Olympus FluoView confocal laser scanning microscope. The internalization of SARS-CoV-2 mimics by cells was examined by flow cytometry analysis (CytoFlex, Beckman Coulter).
Nanodecoys protect lung cells from SARS-CoV-2-mimicking viruses
We studied whether nanodecoys could neutralize SARS-CoV-2 mimic viruses and protect lung cells from being infected. Macrophages and LSCs (1:1) were co-cultured in four-well culture chamber slides, and RhB-labelled lentivirus spike (104 TU mL-1) and DiD-labelled nanodecoys (105) were added. After 2 h of incubation, free RhB-labelled lentivirus spike and DiD-labelled nanodecoys were removed and the samples were washed with PBS three times. Cells were fixed with 4% PFA, stained with LSC (FITC-labelled CD90; 11-0909-42, Invitrogen) or macrophages (CD4) markers, and imaged with an Olympus FluoView confocal laser scanning microscope. Flow cytometry analysis was performed to confirm the microscopy data.
Biodistribution of nanodecoys in mice
All animal procedures were approved by the Institute Animal Care and Use Committee (IACUC) of North Carolina State University (protocol number 19-806-B). Male CD1 mice (aged 7 weeks) were obtained from Charles River Laboratory. DiD-labelled nanodecoys (1 × 1010 particles per kg of body weight) were delivered to the CD1 mice via inhalation treatment using a nebulizer (Pari Trek S Portable Compressor Nebulizer Aerosol System, 047F45-LCS). Mice were killed at 24, 48 and 72 h. All major organs were collected and cryosectioned for further immunofluorescence analysis of the nanodecoys’ in vivo biodistribution post-inhalation.
In vivo clearance of the SARS-CoV-2-mimicking virus by nanodecoys in mice
Before performing the clearance assay, the ACE2 level on the nanodecoys was quantified by ELISA (ab235649, Abcam) and determined to be 112 ACE2 per nanodecoy. AF647-labelled SARS-CoV-2 mimics (5 × 106 per kg of body weight) were first delivered to the male CD1 mice (aged 7 weeks) via inhalation treatment using a nebulizer (Pari Trek S Portable Compressor Nebulizer Aerosol System, 047F45-LCS). Twenty-four hours later, nanodecoys (1 × 1010 particles per kg of body weight) or free rACE2 with the same amount of ACE2 on the nanodecoys were inhaled, respectively. PBS treatment was used as control. Lungs were collected and imaged 1, 2, 3, 4, 5 and 6 d after treatment using Xenogen Live Imager (IVIS). Additionally, lung tissues were cryosectioned for further analysis of the biodistribution of SARS-CoV-2 mimics in vivo post-inhalation. Blood samples were collected for cytokine array analysis (Mouse Cytokine Array C1000, Raybiotech) according to the manufacturer’s instructions.
Toxicity studies in mice
Male CD1 mice (aged 7 weeks) were treated with PBS and LSC- or HEK-nanodecoys (1 × 1010 particles per kg of body weight) via inhalation. After a 14 d treatment, the blood (blood test) and major organs (H&E) were collected for toxicity evaluation.
Non-human primate studies
All animal studies were conducted in compliance with all relevant local, state and federal regulations and were approved by the Bioqual IACUC (number 20-090P). Six cynomolgus macaques (three females, three males) were allocated by a counterbalanced randomization. All animals were housed at Bioqual. The macaques were challenged with SARS-CoV-2 using the intranasal and intratracheal routes. The viral inoculum (0.5 ml) was administered dropwise into each nostril and 1.0 ml of viral inoculum was delivered intratracheally using a French rubber catheter/feeding tube, size 10, sterile (cut 4–6 inches in length). Macaques were inoculated with a total dose of 1.1 × 105 p.f.u. SARS-CoV-2. PBS or the LSC-nanodecoys were administered by inhalation using a nebulizer and fitted mask daily from days 2 to 5 following challenge. BAL, NSs, blood, body weight and body temperature were monitored or collected throughout the study. Macaques were necropsied on day 8 post-challenge. All immunologic and virologic assays were performed blinded.
Statistieken en reproduceerbaarheid
All experiments were performed at least three times independently. Results are shown as means ± SD. Comparisons between any two groups were performed using a two-tailed, unpaired Student’s t test. For multiple group comparison, one-way or two-way analysis of variance (ANOVA) was used with Bonferroni or Tukey post-correction. A P value less than 0.05 was considered statistically significant.
Reporting Summary
Nadere informatie over onderzoeksontwerp is beschikbaar in de Nature Research Reporting Summary gekoppeld aan dit artikel.
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Bron: https://www.nature.com/articles/s41565-021-00923-2
